Molecular genetic studies of control of mRNA stability in the gene for the key enzyme of methionine biosynthesis

高等植物甲硫氨酸生物合成的关键酶--胱硫醚-γ-合成酶(CGS)基因的表达在mRNA稳定阶段受到反馈调节，CGS外显子1的氨基酸序列参与了这一调节。为了确定参与这一调控的因素，构建了一个与CGS基因外显子1融合的绿色荧光蛋白基因(GFP)嵌合基因，并将其置于花椰菜花叶病毒35S启动子的控制下，并将其导入野生型拟南芥。转基因品系GFPc4被用来筛选那些相对于叶绿体自身荧光表现出更高GFP荧光的突变体。这样分离的四个突变体似乎影响了陷阱中CGS的表达。其中之一显示内源CGS和外源GFP融合基因的mRNAs水平升高，而内源CGS和外源GFP融合基因的mRNAs稳定性下调对蛋氨酸应用的响应。以大肠杆菌β-葡萄糖苷酸酶基因为报告基因构建了类似的嵌合基因，并检测了蛋氨酸和S-腺苷蛋氨酸对原生质体表达的影响。结果表明，在突变体中，蛋氨酸和SAM的作用都比亲本有所减弱。SAM已被证明是转录后调控CGS表达的有效因素。结果表明，一个突变体影响了CGS在反式转录后表达的调节。

Expression of the gene for cystathionine gamma-synthase (CGS), the key enzyme of methionine biosynthesis in higher plants is feedback-regulated at the step of mRNA stability and the amino acid sequence in the exon 1 of CGS itself is involved in this regulation. In order to identity the factors that are involved in this regulation, a chimeric gene carrying green fluorescent protein gene (GFP) fused to the exon 1 of CGS gene and placed under the control of a cauliflower mosaic virus 35S promoter was constructed and introduced into wild-type Arabidopsis. The transgenic line termed GFPc4 was used to screen for those mutants that show higher GFP fluorescence relative to the autofluorescence of chloroplasts. Four of the mutants thus isolated seemed to affect the expression of CGS in traps. One of them showed elevated levels of both mRNAs for endogenous CGS and exogenous GFP fusion genes and reduced down-regulation of stability of mRNAs of both endogenous CGS and exogenous GFP fusion genes in response to methionine application. Transient expression analyses by electroporation to the mutant protoplasts were carried out A similar chimeric gene was constructed using E coli beta-glucuronidase gene as a reporter and the effects of methionine and S-adenosylmethionine (SAM) in the culture medium were tested. The results showed that in the mutant, the effects of both methionine and SAM were reduced as compared to the parental line. SAM has been shown to be the efftector of the post-transcriptional regulation of CGS expression. The results suggest that a mutant that affects the post-transcriptional regulation of CGS expression in trans.